1. Technical Field
The present invention is related to the isolation and characterization of a cDNA clone containing the entire coding sequence for the alpha chain of beta-hexosaminidase. More particularly, the present invention is related to a method for either prenatal diagnosing or adult screening for a genetic defect which is associated with Tay-Sachs disease.
2. Description of the Background Art
Tay-Sachs disease is an inherited disorder caused by mutation in the alpha-chain of beta-hexosaminidase A, a lysosomal enzyme composed of two polypeptides designated the alpha and beta chains (Stanbury et al., 1983, Metabolic Basis of Inherited Disease, p. 945; Sandhoff et al., 1984, Neuropediatrics 15 Suppl., 85-92). Deficiency of beta-hexosaminidase A results in storage of its major substrate, GM.sub.2 ganglioside. Progressive accumulation of substrate leads to the characteristic neurodegenerative changes of Tay-Sachs disease patients. The disease is heterogeneous displaying a wide range of severity and age of onset. An early onset and fatal form of the disorder referred to as "classic" Tay-Sachs disease has a ten fold higher gene frequency among Ashkenazi Jews than the general population. A less publicized group having a carrier frequency equal to that of Ashkenazi Jews is a population of non-Jewish French Canadians located in Eastern Quebec (Anderman et al., 1977 Prog. Clin. Biol. Res. 18:161). In terms of age-of-onset, clinical course and biochemical parameters, French Canadian patients are indistinguishable from Ashkenazi patients (Hechtman et al., 1983 Clin. Gen. 24:206).
Beta-Hexosaminidase is a lysosomal enzyme composed of two polypeptide chains, alpha and beta, encoded on different chromosomes. Association of these polypeptides, a prerequisite for catalytic activity, results in three isozymes. Of these, only the A isozyme (aB) is able to hydrolyze all known beta-hexosaminidase substrates (beta-N-acetylglucosaminides and beta-N-acetylgalactosaminides, as well as 6-sulfated beta-N-acetylglucosaminides). The .beta. isozyme (.beta..beta.) is unable to hydrolyze GM.sub.2 ganglioside and the sulfated substrates, whereas the S isozyme (.alpha..alpha.) has little catalytic activity except toward the sulfated compounds (Stanbury et al., 1983, Metabolic Basis of Inherited Disease, p. 945; Sandhoff et al., 1984, Neuropediatrics 15 Suppl., 85-92). Different specificities in substrate binding and hydrolysis have been recently proposed for the alpha and beta chains (Kytzia et al., J. Biol. Chem. 256:7568-7572, 1985).
The biosynthesis of beta-hexosaminidase follows that of a typical lysosomal enzyme (Hasilik et al., 1984, in Lysosomes in Biology and Pathology, 7:pp3-16). Synthesis occurs on membrane-bound polysomes and insertion into the endoplasmic reticulum is followed by cleavage of the signal sequence and glycosylation of the enzyme. In the Golgi apparatus, the lysosomal enzymes are selected from the melange of glycoproteins for phosphorylation of certain mannose residues to form the recognition marker for binding to phosphomannosyl receptor and subsequent transport to the lysosomes. Within the lysosome, beta-hexosaminidase undergoes proteolytic processing from precursor to mature form. Association of the alpha and beta chains occurs after phosphorylation and is therefore thought to take place in the Golgi apparatus (Proia et al, J. Biol. Chem. 259:3350-3354, 1984).
Mutations in the genes encoding the alpha or beta chain of beta-hexosaminidase result in Tay-Sachs or Sandhoff disease, respectively--genetic disorders displaying both clinical and biochemical heterogeneity (Sandhoff et al., 1984, Neuropediatrics 15 Suppl., 85-92). The best known of these is the classic late-infantile form of Tay-Sachs disease that occurs among Jews of Ashkenazi origin. A recent study reported the isolation of a clone containing a cDNA fragment for the alpha chain of human beta-hexosaminidase and a deficiency of mRNA in Ashkenazi Tay-Sachs fibroblasts (Myerowitz et al., 1984, PNAS, USA, 81:5394). However, the isolation and characterization of a cDNA clone containing the entire coding sequence of the alpha chain of human beta-hexosaminidase, has not heretofore been achieved.
In order to detect the many different kinds of mutations in human alpha-chain beta-hexosaminidase genes in individuals clinically displaying Tay-Sachs disease, one must have the entire, full length cDNA. If not, one would miss those mutations occuring in that part of the gene for which there is no cDNA. For example, the French-Canadian mutation occurs at the 5'-end of the gene which the prior cDNA clone (Myerowitz et al. PNAS, 1984 supra) did not have. Therefore, it is quite evident that the prior clone would have never made it possible to detect and elucidate the nature of Tay-Sachs mutation, for example, in the non-Jewish French-Canadian population.